Human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and the technology to developmentally program these cells to various cell lineages offer great promise for cell therapy of various diseases. Recently, different protocols to differentiate them into hepatocytes-like cells (HLC) have been described. In this context, we hypothesize that this approach could be useful in developing a relevant model for HCV infection in vitro. Moreover, the hiPSCs approach could allow the generation of patient-specific hepatocytes with promising opportunity for cell therapy of viral liver diseases. We have generated hiPSCs from primary fibroblasts using lentiviruses or Sendai virus vectors, and characterized them in comparison to hESCs. Human pluripotent stem cells were efficiently differentiated into HLC, as demonstrated by induction of the expression of hepatic markers and the secretion of hepatic proteins (AFP and albumin) in the supernatants. Moreover these cells recapitulate hepatocyte-specific metabolic functions like lipid and glycogen accumulation, and indocyanin green metabolism. These HLC can be infected with HCV. The infected cells also respond to antiviral therapy, such as interferon-alpha and 2'-methylcytidine, a nucleoside analog inhibitor of HCV polymerase. HCV infection also induces intrinsic innate immune response including interferon response. To show whether it is possible to successfully engraft these cells and establish functional human hepatocytes in vivo, we engrafted, via intra-splenic injection, 2-4 millions HLCs into the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter (MUP-uPA/SCID/Bg). Human albumin (hALB) could be detected in the serum of the engrafted mice by ELISA as early as day 10 post-engraftment, with concentrations ranging from 0.4 to 2.3 mg/mL. More importantly, hALB persisted for more than 4 months, consistent with long-term engraftment of human cells in the mouse liver parenchyma. Mice were sacrificed 4 months post-engraftment, and liver sections were assessed by immunostaining for a variety of human proteins (albumin, alpha-1-antitrypsine, alpha-fetoprotein). Areas of human cells were observed around central veins, and could constituted up to 15% of the mouse liver parenchyma. 2 weeks post-engraftment, mice with high hALB concentration were inoculated with HCV positive sera of different genotypes (1a, 1b, 3). Serum samples were obtained at day 30, 60 and 90 post inoculation, and assessed for HCV RNA by RTqPCR. HCV RNA could be detected in the serum of every mouse at day 60 post-inoculation. HCV increased up to 90 days post-infection, consistent with long-term infection of engrafted human hepatocytes in the mouse liver. We demonstrate here that hESCs- and hiPSCs-derived DHHs can be efficiently engrafted into the mouse liver parenchyma, and that they can be infected by HCV(+) sera of different genotypes. This approach constitutes a valuable model to study HCV infection in the context of patients genetic background as well as in the native architecture of the liver. In collaboration with Dr. Cindy Dunbar's lab, we have also extended the hepatic differentiation protocol to monkey iPSC lines. The goal is to establish a monkey model for potential stem cell-based therapy. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. We have developed a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain highly differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSCs-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli. HCV infection in primary human hepatocyte (PHH) can induce robust innate immunity thus mimicking HCV infection in vivo as previously described in our lab. In an effort to develop new model systems to study HCV infection, we explored single-cell technologies to investigate the host transcriptional responses of PHH upon HCV infection. We used flow cytometry to sort more than 1,200 single cells from HCV-infected PHH populations at different time points. We then applied the single-cell gene expression assay to systematicaly profile the HCV-infected, bystander and mock-infected cells by quantified 96 genes including HCV-associated host factors, innate immunity-associated genes, different types of interferons, interferon-stimulated genes (ISGs), inflammatory cytokines and chemokines, and lipid metabolism genes. We found that IL28, a type III interferon, was significantly up-regulated in HCV-infected cells compared to bystander cells. Importantly, most ISGs were readily detected in HCV-infected cells argueing against viral protein directly interfering with innate immune signaling in infected hepatocytes. In addition, we observed that the levels of HCV RNA, IL28 and ISGs expression were positively correlated at the single-cell level indicating that HCV replication drives the interferon response and type III interferons are the predominant mediator of this response in the infected hepatocytes. Interestingly, by comparing single cell dataset from HCV-infected to bystander cells, we identified a group of infected cells with unique signatures that may be implicated in effective antiviral control. Finally, we find extensive, and previously unobserved, bimodal variations in ISG mRNA expression that may modulate viral persistence and interferon signaling circuits. Our study demonstrates the power of single-cell expression profiling as a novel model system to study the impact of heterogeneous host-pathogen interactions in disease progression and may provide new insight for antiviral therapies.